Extracts from the fruits of milk thistle (Silybum marianum (L.) Gaertn.) are known by their contents of the flavanolignan-type substances having polyhydroxyphenyl chromanone skeleton (G. Hahn et al., Arzneimittel—Forschung Drug Res. 18, 698-704, (1968)). These polyhydroxyphenyl chromanones comprise silybin and its enantiomers (silybin A, silybin B, isosilybin A, isosilybin B), silydianin, and silychristin that are altogether referred to as a powder extract of Silybum marianum or also as silymarin. The extract of Silybum marianum, CAS No: [84604-20-6] is almost insoluble in water, it is soluble in acetone, ethyl acetate, methanol, and ethanol. Qualitative requirements for the extract are defined, e.g., by the pharmacopoeia standards USP/NF or Ph Eur.
In practice also silybin, CAS No. [22888-70-6], chemical name 3,5,7-trihydroxy-2-[3-(4′-hydroxy-3′-methoxyphenyl)-2-hydroxymethyl)-1,4-benzodioxan-6-yl]-4-chromanone, of the molecular formula of C25H22O10 and molecular weight 482.443, is used. The melting point of commercially available silybin is about 158° C. for anhydrous substance and about 167° C. for monohydrate.
However, all the herein above mentioned substances are very little soluble in aqueous media.
The solubility of silybin at 25° C. (mg/ml) in solvents miscible with water is as follows:
Ethoxy diglycol 350.1 ± 10.4Polyethylene glycol 200345.9 ± 9.5Polyethylene glycol 400/ethanol (1:1)342.1 ± 7.1Ethanol225.2 ± 5.2Propylene glycol162.4 ± 3.6Water 0.4 ± 0.1
The solubility of silybin at 25° C. (mg/ml) in solvents not fully miscible with water is as follows:
Glyceryl monooleate33.2 ± 2.8 Tocopherol20.0 ± 1.9 Castor oil7.1 ± 1.2Ethyl linoleate2.1 ± 0.8Capryl-caprine triglyceride0.8 ± 0.5Fish oil0.5 ± 0.2
The silybin solubilities are given according to: Jong Soo Woo, Tae-Seo Kim, Jae-Hyun Park, and Sang-Cheol Chi: Formulation and Biopharmaceutical Evaluation of Silymarin Using SMEDDS. Arch Pharm Res Vol. 30, No 1, 82-89, 2007.
The phenolic nature of flavanolignans is given by the substitution by two hydroxyl groups in positions 5 and 7 on the 4-chromanone skeleton and by one hydroxyl group in position 4′ on the side hydroxyl-methoxyphenyl group. Their dissociation constant pK values are as follows:
silybinpKa.1[25° C.] = 7.00; pKa.1[37° C.] = 6.86pKa.2[25° C.] = 8.77; pKa.2[37° C.] = 8.77pKa.3[25° C.] = 9.57; pKa.3[37° C.] = 9.62silychristinpKa.1[25° C.] = 6.52; pKa.1[37° C.] = 6.62pKa.2[25° C.] = 7.22; pKa.2[37° C.] = 7.41pKa.3[25° C.] = 8.96; pKa.3[37° C.] = 8.94silydianinpKa.1[25° C.] = 6.64; pKa.1[37° C.] = 7.10pKa.2[25° C.] = 7.78; pKa.2[37° C.] = 8.93pKa.3[25° C.] = 9.66; pKa.3[37° C.] = 10.06
The pK value data are quoted according to: Meloun, Milan; Syrový, Tomá{hacek over (s)}; Bordovská, Sylva; Vrána, Ale{hacek over (s)}: Reliability and uncertainty in the estimation of pKa by least squares nonlinear regression analysis of multiwavelength spectrophotometric pH titration data. Analytical and Bioanalytical Chemistry, 2007, 387 (3), 941-955.
All substances mentioned above exhibit a broad spectrum of pharmacologic activities: antioxidant properties, stabilization of cell membranes, stimulation of protein biosyntheses. These effects constitute the basis for utilization of silymarin and its components, particularly silybin (a mixture of silybins A and B and even isosilybins A and B) as a hepatoprotective agent. Silymarin acts against both acute and chronic liver intoxication by many toxins, demonstrably by carbon tetrachloride, galactose amine, paracetamol, ethanol, phalloidin, and α-amanitin. Silymarin is thus an active component of many preparations used for the therapy and prevention of liver diseases (LEGALON®, FLAVOBION®, etc.). More information on recent research on silybin and silymarin and their use can be found in survey paper by K{hacek over (r)}en V., Walterová D.: Silybin and silymarin—new effects on applications. Biomed. Papers 149 (1), 29-41, 2005.
The powder extract of Silybum marianum used for the preparation of hepatoprotective pharmaceutical preparations or food additives is a refined extract with the standardized content of flavanolignans. According to USP/NF it should contain 40-80% of flavanolignans sum, evaluated by liquid chromatography to silybin standard. According to the recent studies, the therapeutic potential of the Silybum marianum substances can be used, along to the traditional hepatoprotective effects, particularly for prophylactic enhancement of the health condition of people endangered by diabetes and colon cancer.
Experimental studies on hypoglemic effects of Silybum marianum (Males R. J., Farnsworth N. R.: Antidiabetic plants and their active constituents. Phytomedicine 2 (2), 137-189, 1995) and on positive effects of flavanolignanes on diabetes (Soto C. P. et al.: C. Pharmacology, Toxicology & Endocrinology, 119 (2), 125-129, 1998) have been published.
Use of flavanolignans from Silybum marianum as adjuvants in the chemotherapy of tumours (U.S. Pat. No. 5,714,473) and as antiproliferative medicaments (U.S. Pat. No. 5,912,265) has been recently protected by patents.
Silymarin in the form of silymarin methylglucamine is also proposed as a component of preparations against the effects of alcohol (U.S. Pat. Nos. 6,913,769 a 6,967,031).
Use of the flavanolignans of Silybum marianum in medicaments as well as in other applications in which they affect particularly the sensoric properties of products such as their taste and colour is limited by their low solubility in both hydrophilic and lipophilic solvents.
A number of approaches have been proposed for overcoming this insufficient solubility such as, e.g., by the preparation of silymarin component salts with N-methylglucamine (U.S. Pat. No. 3,994,925). These salts with monoamino polyhydroxyalkyl alcohols hydrolyze easily, thus they require stabilization with a substantial amount of polyvinyl pyrrolidone or albumin. Similarly, a procedure comprising preparing the solution of silymarin flavanolignans in polyvinyl pyrrolidone and lyophilising this solution has also been proposed (U.S. Pat. No. 4,081,529).
Esters of silybin with dicarboxylic acids, e.g., with disodium salt of bis-hemisuccinate of silybin according to U.S. Pat. No. 5,196,448, are destined for dermatologic and cosmetic applications in topic application forms.
The preparation of silybin glycosides that are more soluble in water than silybin and exhibit similar effects as silymarin is protected by the CZ Patent 287 657. However, the industrial production of glycosides is technologically demanding and costly.
Complex compounds of silymarin or silybin with phospholipids, described in U.S. Pat. Nos. 4,764,508 and 4,895,839, are prepared by dissolving the components (1 mol of silymarin or silybin and 0.3 to 2.0 mol of phosphatidyl choline, phosphatidyl serine, or phosphatidyl ethanol amine) in an aprotic solvent (dioxane or acetone) and by precipitating the complex by adding an aliphatic hydrocarbon or by lyophilisation or spray drying. In addition to the ecologically undesirable utilization of organic solvents, the resulting complex compounds form disperse systems in contact with water. This feature orientates the application of these complex compounds more to the topic preparations.
Inclusion complexes of silybin with cyclodextrins are described in U.S. Pat. No. 5,198,430. Complexes of silybin with α-, β- and γ-cyclodextrin and their derivatives in molar ratio 1 mol of silybin to 1 to 4 mol of the respective cyclodextrin have been described. The complexes are prepared by dissolving the two components in aqueous ammonia and by removal of ammonia either by evaporation or neutralization by hydrochloric acid and by subsequent drying or lyophilisation. For the preparation of these inclusion complexes physiologically unsuitable ketonic solvents such as acetone, cyclohexanone, methylisobutyl ketone, and diethyl ketone are used, the residual amounts of which in the complexes must be monitored and removed. The thus prepared complexes exhibit a better biological availability than the flavanolignans themselves, however, their solubility in water is enhanced but very little so that anion-active tensides should be used in drug forms.
A procedure based on dissolving the flavanolignanes of Silybum marianum in an alkanolic or ketonic solvent and mixing this solution with an auxiliary substance from the group of tetritols, pentitols, hexitols, or vinylpyrrolidonic or ethylene oxide homopolymers and the subsequent removal of the solvents (CZ Patent 292 832) prevents the formation of crystalline forms causing the low solubility. The solid drug forms prepared from the thus obtained substances exhibit better dissolution parameters, but they are not water soluble in the exact meaning of the word.
Processes for enhancing the bioavailability of silymarin based on the preparation of co-precipitates of flavanolignans with carriers and detergents are covered by a patent (U.S. Pat. No. 5,906,991). Water-soluble saccharides, derivatives of cellulose, and polyvinylpyrrolidon are described as suitable carriers, polysorbates of fat acids are used as detergents. In the dissolution test these co-precipitates exhibit a higher solubility than the non-modified silymarin so that their higher bioavailibility can be expected.
The so far known water-soluble forms of the Silybum marianum extract are not fully suitable for application in food industry, e.g., for use in beverages like beer, beverages on the basis of beer, and soft drinks, as some of their components are not approved for use in food industry.